Chemical constituents from Terminalia glabrescens

Abstracts

A new oleanane-type triterpene (3beta,6beta,23,28-tetrahydroxyolean-12-ene) was isolated from the leaves of Terminalia glabrescens, together with ursolic, 2alpha-hydroxyursolic, oleanolic, maslinic, arjunolic, sumaresinolic and asiatic acids, squalene, phytol, sitosterol-3-O-beta-D-glucopyranoside and n-alkanes. Friedelin, taraxerol, lupeol, lupenone, betulin, betulone, betulinic acid, arjunglucoside I, stigmastane-3beta,6alpha-diol, beta-sitosterol, (-) catechin,beta-D-pyranotagatose, beta-D-furanofructose and alpha-D-furanofructose were obtained from the trunk bark.

Terminalia glabrescens; Combretaceae; triterpenes; 3beta,6beta,23,28-tetrahydroxyolean-12-ene; stigmastane-3beta,6alpha-diol

Das folhas de Terminalia glabrescens foram obtidos um novo triterpeno pentacíclico (3beta,6beta,23,28-tetraidroxiolean-12-eno), além dos ácidos ursólico, 2alfa-hidroxiursólico, oleanólico, maslínico, arjunólico, sumaresinólico e asiático, esqualeno, fitol, sitosterol-3-O-beta-D-glucopiranosídeo e n-alcanos. Da casca do caule foram obtidos friedelina, taraxerol, lupeol, lupenona, betulina, betulona, ácido betulínico, arjunglucosídeo I, estigmastano-3beta,6alfa-diol, beta-sitosterol, (-) catequina, beta-D-piranotagatose, beta-D-furanofrutose e alfa-D-furanofrutose.

SHORT REPORT

Chemical constituents from Terminalia glabrescens

Fernanda R. Garcez^*; Walmir S. Garcez; Daniel L. S. Miguel; Alessandro A. T. Serea; Fabiana C. Prado

Departamento de Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal de Mato Grosso do Sul, 79070-900 Campo Grande - MS, Brazil

ABSTRACT

A new oleanane-type triterpene (3b,6b,23,28-tetrahydroxyolean-12-ene) was isolated from the leaves of Terminalia glabrescens, together with ursolic, 2a-hydroxyursolic, oleanolic, maslinic, arjunolic, sumaresinolic and asiatic acids, squalene, phytol, sitosterol-3-O-b-D-glucopyranoside and n-alkanes. Friedelin, taraxerol, lupeol, lupenone, betulin, betulone, betulinic acid, arjunglucoside I, stigmastane-3b,6a-diol, b-sitosterol, (-) catechin, b-D-pyranotagatose, b-D-furanofructose and a-D-furanofructose were obtained from the trunk bark.

Keywords: Terminalia glabrescens, Combretaceae, triterpenes, 3b,6b,23,28-tetrahydroxyolean-12-ene, stigmastane-3b,6a-diol

RESUMO

Das folhas de Terminalia glabrescens foram obtidos um novo triterpeno pentacíclico (3b,6b,23,28-tetraidroxiolean-12-eno), além dos ácidos ursólico, 2a-hidroxiursólico, oleanólico, maslínico, arjunólico, sumaresinólico e asiático, esqualeno, fitol, sitosterol-3-O-b-D-glucopiranosídeo e n-alcanos. Da casca do caule foram obtidos friedelina, taraxerol, lupeol, lupenona, betulina, betulona, ácido betulínico, arjunglucosídeo I, estigmastano-3b,6a-diol, b-sitosterol, (-) catequina, b-D-piranotagatose, b-D-furanofrutose e a-D-furanofrutose.

Introduction

Plants of the genus Terminalia (Combretaceae) are known as a rich source of secondary metabolites, such as pentacyclic triterpenes and their glycoside derivatives, flavonoids, tannins and other aromatic compounds, some of which with antibacterial, antifungal, anticancer and hepatoprotective activities.^1-8

Terminalia glabrescens Mart., which has not previously been chemically investigated, is a medium-sized tree widespread in Mato Grosso do Sul, Brazil. In the present paper, we describe the isolation and structural elucidation of a new oleanane-type triterpene (3b,6b,23,28-tetrahydroxyolean-12-ene) from the leaves of this species. The known pentacyclic triterpenes ursolic, 2a-hydroxyursolic, oleanolic, maslinic, arjunolic, sumaresinolic and asiatic acids together with squalene, phytol, sitosterol-3-O-b-D-glucopyranoside and foliar wax hydrocarbons were also obtained from the leaves, while the trunk bark afforded the triterpenes friedelin, taraxerol, lupeol, lupenone, betulin, betulone, betulinic acid and arjunglucoside I in addition to stigmastane-3b,6a-diol, b-sitosterol, (-) catechin, b-D-pyranotagatose, b-D-furanofructose and a-D-furanofructose.

The structures of the known and new compounds were established on the basis of spectral data, mainly ¹H and ¹³C (1D and 2D) NMR spectra and by comparison with authentic samples.

Results and Discussion

The hexane and CHCl₃ solubles obtained from partition of the ethanol extract of leaves were subjected to a series of normal and reversed phase silica gel column chromatography, gel filtration and preparative TLC on silica gel separations to yield the new pentacyclic triterpene 3b,6b,23,28-tetrahydroxyolean-12-ene (1) in addition to ursolic,⁹ oleanolic,⁹ 2a-hydroxyursolic,⁹ maslinic,⁹ sumaresinolic (2),⁹ asiatic (3)⁹ and arjunolic (4)⁹ acids, squalene,¹⁰ phytol, sitosterol-3-O-b-D-glucopyranoside and long chain hydrocarbons. These were characterized as n-alkanes in the range between C₁₈ and C₃₃, with a large predominance of chains with odd numbers of carbon atoms, where C₂₉ and C₃₁ were found as the main homologues. The isolation of squalene and of the triterpene 2 in the genus Terminalia is being reported for the first time. The known compounds were identified by their ¹H and ¹³C NMR spectral data, by comparison with literature values and/or with authentic samples. Identification of 2 as well as the isomeric triterpenes maslinic / 2a-hydroxyursolic acids and 3 / 4 was supported by conversion into their corresponding C-28 methyl ester derivatives whose ¹H and ¹³C NMR resonances were in accordance with reported data.^9,11 The alkane composition was determined on the basis of GC-FID retention times and by comparison with authentic standards.

Compound 1 was obtained as an amorphous solid and its HBBD ¹³C NMR spectrum displayed signals for 30 carbon atoms. With the aid of information afforded by the DEPT spectra these signals could be attributed to seven quaternary, six methine, eleven methylene and six methyl carbon atoms. The presence of a trisubstituted double bond was inferred by the signals of a methine carbon at d 123.0 and a quaternary carbon at d 144.4. In the HMQC spectrum a cross-peak correlation was observed between the former carbon signal and the broad hydrogen singlet at d 5.57, which was assigned to the vinylic hydrogen. In the ¹H NMR spectrum, the signals at d 5.04 (br s) and 4.26 (dd, J 11.4 and 4.1 Hz) which showed connectivities in the HMQC spectrum with the carbon signals at d 67.7 and 73.4, respectively, were attributed to two carbinolic hydrogens. Similarly, the broad singlet at d_H 4.06 (2H) and the two doublets at d_H 4.38 (1H, J 10.5 Hz) and 4.03 (1H, J 10.5 Hz), which showed cross-peak correlations with the carbon signals at d 64.5 and 67.2, respectively, were assigned to hydroxymethylene hydrogens. These information, along with the absorption at n_max 3429 cm ^–1 observed in the IR spectrum, led to the assumption that 1 was an olean-12-ene-type triterpene with two hydroxymethylene and two secondary hydroxyl groups and its molecular formula established as C₃₀H₅₀O₄. The aforementioned data when compared with those of other known structurally related compounds suggested that 1 would have the same functionality on rings A and B as 3b, 6b, 23-trihydroxyolean-12-en-28-oic acid (5) previously isolated from Timonius timon (Rubiaceae).¹² Indeed, the ¹H and ¹³C NMR spectra of 1 showed close resemblance with those of 5, except for the signals due to the carboxylic group at C-28 observed in the spectra of the latter, which were replaced by a singlet at d_H 4.06 (2H), indicative of a hydroxymethylene group at C-28. Unambiguous assignments of the hydroxymethine carbons C-3 and C-6 (d 73.4 and 67.7, respectively) were established on the basis of connectivities observed from an HMBC experiment (Table 1). Accordingly, cross-peak correlations between the carbon signals of C-23 and C-24 and H-3 resonance at d 4.26, which in turn displayed one-bond ¹H-¹³C connectivity with the carbon signal at d 73.4 allowed the assignments of C-3/H-3. In a similar fashion, H-6 (d 5.04) presented long-range correlations with C-7, C-8 and C-10. The appearance of H-6 as a broad singlet in the ¹H NMR spectrum indicated its a-equatorial orientation. A similar feature was also observed for the signal of H-6 in sumaresinolic acid methyl ester 2a [d_H 4.53 (br s)] and in 6b-hydroxymaslinic acid [d_H 4.85 (s)]¹³ which bear the same stereochemistry as H-6 in 1 and 5. The b-hydroxyl substitution at C-3 was inferred by the chemical shift and multiplicity of the axial H-3 observed as a double doublet at d 4.26 (J 11.4 and 4.1 Hz). Thus, compound 1 was characterized as 3b,6b,23,28-tetrahydroxyolean-12-ene. Further evidence for the structure of 1 was provided by additional two- and three-bond correlations discernible in the HMBC spectrum (Table 1). After acquisition of its spectroscopic data and storage at room temperature, however, compound 1 was decomposed to a mixture of oxidation products, as revealed by TLC and IR spectroscopy. This fact prevented further analysis of 1 by ESIMS.

After a series of column chromatography separations on silica gel of the hexane and CHCl₃ solubles, obtained from partition of the ethanol extract from the trunk bark, seven triterpenes were isolated, together with stigmastane-3b-6a-diol (7), b-sitosterol, (-) catechin, b-D-tagatose¹⁴ and a- and b-D-fructose.¹⁴ The structures of these triterpenes have been established as friedelin,⁹ lupenone,⁹ lupeol,⁹ betulone,¹⁵ betulin,⁹ betulinic acid⁹ and taraxerol,^9,16 on the basis of spectral analyses and by comparison with previously reported data. In spite of the wide distribution of these compounds in other plant genera, only few records are available for the presence of friedelan- and lupane-type triterpenoids in Terminalia, which is well known for the occurrence of triterpenes with oleanane and ursane skeletons.^8,9 On the other hand, no records related to the isolation of taraxarane-type triterpenes, e.g. taraxerol, have hitherto been reported in this genus.

Compound 6 was identified by means of ¹H and ¹³C NMR as arjunglucoside I, a triterpene glucoside previously characterized in several species of Terminalia (e.g., T. arjuna¹⁷ and T. bellerica¹⁸).

The structure of 7 was shown to be of stigmastane-3b-6a-diol on the basis of its ¹H and ¹³C NMR spectral data, which were in accordance with those reported for the same steroid previously isolated from Trichosantes kirilowii (Cucurbitaceae),¹⁹Spatholobus suberetus (Leguminosae)²⁰ and Urtica dioica (Urticaceae)²¹ and until now, not yet described in Combretaceae.

b -sitosterol, sitosterol-3-O-b-D-glucopyranoside and (-) catechin were identified by comparison with authentic samples.

Experimental

General experimental procedures

¹H and ¹³C, ¹H-¹H COSY, HMQC and HMBC NMR spectra were recorded on a Bruker DPX-300 spectrometer. Standard pulse sequences were used for homo- and heteronuclear correlation experiments. FT-IR spectra were obtained on KBr pellets in a Bomem MB-100 spectrometer. Optical rotations were measured with a Perkin-Elmer 341 polarimeter. GC analysis of the n-alkanes was performed on a Shimadzu QP-5000 GC, using a capillary column (LM-5, 30m x 0.25 mm), FID, He as carrier gas, a temperature program from 80^o to 300 ^oC at 5^o min ^-1 and Sigma n-alkane standards. Silica gel 60 70-230 and 230-400 mesh, RP-18 silica gel 230-400 mesh and Sephadex LH-20 were used for column chromatography. Preparative TLC was performed on silica gel 60 PF₂₅₄ plates.

Plant material

The leaves and trunk bark of Terminalia glabrescens Mart. were collected in Campo Grande, Mato Grosso do Sul, Brazil, in June, 1996. The plant was identified by Dr. Nilda Marquette (Jardim Botânico do Rio de Janeiro, RJ, Brazil) and a voucher specimen, 11264, is deposited in the Herbarium of the Universidade Federal de Mato Grosso do Sul.

Extraction and isolation of chemical constituents

Air-dried and powdered leaves (1.9 kg) were extracted at room temperature with EtOH. The residue obtained from the EtOH extract was subsequently partitioned between EtOH-H₂O (9:1) and hexane and EtOH-H₂O (1:1) and CHCl₃. The hexane phase (11.9 g) was subjected to CC on silica gel (70-230 mesh), eluted with a gradient of hexane-CHCl₃-EtOAc-MeOH resulting in 43 frs. of 125 mL each. Fraction 1 consisted of a mixture (38 mg) of C₁₈ - C₃₃n-alkanes, while fraction 2 yielded squalene (48 mg).

Fractions 16-17 afforded ursolic acid (10 mg) and a mixture (5 mg) of ursolic and oleanolic acids. Further amounts of the former (15 mg) were obtained from fr. 19.

Fractions 20 and 33-39 yielded, respectively, a mixture of 2a-hydroxyursolic and maslinic acids (5 mg) and sitosterol-3-O-b-D-glucopyranoside (5 mg).

The CHCl₃ phase (16.8 g) upon CC over silica gel (70-230 mesh) eluted with a gradient of hexane-CHCl₃-EtOAc-MeOH afforded 37 fractions of 125 mL each. Fractions 5-7 and 21-22 consisted of phytol (23 mg) and ursolic acid (43 mg), respectively.

Fraction 25 was separated by CC on RP-18 silica gel, eluting with CHCl₃-MeOH-H₂O (2.0:4.0:1.5 to pure MeOH) to yield 11 main fractions A®K (20 mL each). CC on silica gel (230-400 mesh) of fraction C eluted with CHCl₃-MeOH (12.0:0.5) yielded further 29 fractions (10 mL each). From these, fractions 26-27 consisted of a mixture of 2a-hydroxyursolic acid, maslinic acid and 2 and was treated with an ethereal solution of diazomethane to give, after prep. TLC on silica gel [hexane-CHCl₃-MeOH (3:6:1)], 2a-hydroxyursolic acid methyl ester (7 mg) and a mixture (19 mg) of methyl esters of sumaresinolic 2a and maslinic acids.

Prep TLC on silica gel [CHCl₃-isopropanol (12:2)] of fraction 28 yielded 1 (10 mg).

Fraction 31 was further separated on a RP-18 silica gel (230-400 mesh) using CHCl₃-MeOH-H₂O (2.0:4.0:1.5 to pure MeOH) as eluent to give 5 main fractions (A®E). Fraction B, after treatment with Et₂O/CH₂N₂ followed by prep. TLC on silica gel [CHCl₃-MeOH (10.0:0.5)] gave 2a (19 mg) and a mixture of methyl esters of 3 and 4 (40 mg). The same methylation and separation procedures applied to fr. C yielded 2-a-hydroxyursolic acid methyl ester (18 mg) and further amounts of a mixture of methyl esters of 3 and 4 (10 mg). Fractions D and E consisted of 2-a-hydroxyursolic acid (36 mg) and sitosterol-3-O-b-D-glucopyranoside (20 mg), respectively.

Air-dried and powdered trunk bark (1 kg) was extracted with EtOH at room temperature to obtain an EtOH extract which, after concentration under reduced pressure, was partitioned successively between MeOH-H₂O (9:1) and hexane and between MeOH–H₂O (1:1) and CHCl₃.

The hexane phase (4.5 g) was chromatographed on a silica gel (70-230 mesh) column eluted with increasing amounts of EtOAc in hexane to obtain 35 fractions of 100 mL each.

Fractions 5 and 6 afforded, respectively, friedelin (15 mg) and a mixture of friedelin and lupenone (20 mg).

Fraction 7 was treated with hot benzene. The insoluble residue was then washed with acetone to yield a precipitate and a supernatant fraction. The former was further chromatographed on a silica gel (230-400 mesh) column, with a gradient of hexane-EtOAc to give friedelin (25 mg) and taraxerol (29 mg). In a similar fashion, the acetone-soluble fraction afforded lupeol (10 mg) and 7 ( 8 mg).

From fraction 11 was obtained b-sitosterol (15 mg).

The CHCl₃ phase (18.0 g) was fractionated by CC on silica gel (70-230 mesh) eluted with a gradient of hexane-EtOAc-MeOH leading to 76 fractions (125 mL each). Fractions 10-11, 12-16, 17 and 58-59 yielded betulone (13 mg), betulin (10 mg) betulinic acid (12 mg) and (-) catechin (10 mg), respectively. Fractions 69 and 71 gave, respectively, 6 (18 mg) and an unresolved mixture (16 mg) of b-D-tagatose and a- and b-D-fructose.

3b,6b,23,28-tetrahydroxyolean-12-ene (1). Colorless amorphous solid. [a]_D²³: + 29.9^o (MeOH; c 0.43). IR n_max/cm^-1: 3429, 2940, 1457, 1035 (KBr). ¹H and ¹³C NMR (Table 1).

Stigmastane-3b,6a-diol (7). ¹H NMR (300 MHz, CDCl₃): d 3.56 (tt, J 11.0; 4.5 Hz, H-3), 3.40 (dt, J 4.5; 10.7 Hz; H-6), 0.63 (s, CH₃-18), 0.79 (s, CH₃-19), 0.88 (d, J 6.4 Hz, CH₃-21), 0.81 (d, J 6.6 Hz, CH₃-26), 0.81 (d, J 6.6 Hz, CH₃-27), 0.80 (t, J 7.8 Hz, CH₃-29). ¹³C NMR (75 MHz, CDCl₃): d 37.2 (C-1), 31.0 (C-2), 71.3 (C-3), 32.2 (C-4), 51.7 (C-5), 69.5 (C-6), 41.7 (C-7), 34.3 (C-8), 53.8 (C-9), 36.3 (C-10), 21.1 (C-11), 39.8 (C-12), 42.6 (C-13), 56.2 (C-14), 24.2 (C-15), 28.2 (C-16), 56.1 (C-17), 12.0 (C-18), 13.4 (C-19), 36.1 (C-20), 18.7 (C-21), 33.9 (C-22), 26.0 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.0 (C-27), 23.0 (C-28),12.0 (C-29).

Acknowledgements

The authors are grateful to CPq-PROPP-UFMS and PROAP-CAPES for financial support and to CNPq for the award of fellowships. Thanks are also due to Dr. Nilva R. Poppi and Luiz Leonardo S. Viana (Departamento de Química, UFMS) for GC analysis of the n-alkanes.

Received: September 24, 2002

Published on the web: April 11, 2003

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Publication Dates

• Publication in this collection
  16 June 2003
• Date of issue
  May 2003

History

• Accepted
  11 Apr 2003
• Received
  24 Sept 2002